This invention relates generally to the field of gene sequencing. More particularly, this invention relates to a gene sequencer, a high density bio-compact disk useful therewith and a method of sample preparation therefor. The high-density bio-compact disk and the sample preparation methodology find application in the field of oligonucleotide sequencing and DNA sequencing and detection generally.
In one aspect, the present invention features a sample preparation method for obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) forming a solid support having all possible n-mer oligonucleotides attached to the surface of the support; (b) contacting the solid support resulting from step (a) with the sample under conditions causing the sample oligonucleotides to hybridize with the complementary n-mer oligonucleotides on the solid support; (c) contacting the solid support resulting from step (b) with a hydrolyzing agent; (d) separating the unbound oligonucleotides from the hybridized oligonucleotides; and (e) denaturing the hybridized n-mer oligonucleotides to obtain the n-mer oligonucleotides of the sample; wherein n is an integer selected from the integers 4-10,000, most advantageously 6-28.
In another aspect, the invention features a method of obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) contacting a solid support adapted to couple with oligonucleotides in the sample with at least a portion of the sample; (b) contacting the solid support resulting from step (a) with a mixture of n-mer oligonucleotides for a time sufficient for the n-mer oligonucleotides to hybridize with the complementary n-mer oligonucleotides on the solid support; (c) separating the hybridized n-mer oligonucleotides from the unhybridized oligonucleotides; (d) denaturing the hybridized n-mer oligonucleotides to obtain the n-mer oligonucleotides complementary to those present in the sample; wherein n is an integer selected from the integers 4-10,000, most advantageously 6-28.
In still another aspect, the sample preparation method includes a method of obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) contacting a solid support having bound thereon oligonucleotides from a sample with a mixture of a plurality of oligonucleotides having (k+m)-mers, wherein k+m=n, with a mixture of a plurality of first oligonucleotides, each being a k-mer and being without a free hydroxyl group at the 3xe2x80x2-end thereof, and a plurality of second oligonucleotides, each being a m-mer and being without a free phosphate group at the 5xe2x80x2-end thereof; (b) ligating the oligonucleotides on the solid support resulting from step (a); (c) removing the unligated oligonucleotides from the solid support; and (d) denaturing the hybridized n-mer oligonucleotides remaining on the solid support to obtain the n-mer oligonucleotides complementary with those present in the sample; wherein m, k and n are each an integer selected from the integers from 6-10,000, most advantageously 12-40, with the proviso that k+m=n.
In yet another aspect, the sample preparation method includes a method of obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) contacting a solid support having bound thereon a plurality of oligonucleotides from a sample with a mixture of a plurality of h-mer oligonucleotides each having a phosphate group at both the 3xe2x80x2- and 5xe2x80x2-end, a plurality of i-mer oligonucleotides each having a hydroxyl, amino or thiol group at the 3xe2x80x2-end and no terminal phosphate group, and a plurality of j-mer oligonucleotides having a hydroxyl, amino or thiol group at the 5xe2x80x2-end and no terminal phosphate group; (b) chemically or enzymatically ligating the oligonucleotides on the solid support resulting from step (a); (c) removing the unligated oligonucleotides from the solid support resulting from step (b); and (d) denaturing the hybridized n-mer oligonucleotides remaining on the solid support to obtain the n-mer nucleotides complementary with those present in the sample; wherein h, i and j are each an integer selected from the integers from 6-10,000, most advantageously 18-60, with the proviso that h+i+j=n.
In yet another aspect of this invention, an assay element is described comprising a substrate having a surface including a plurality of discrete areas on the surface adapted to attach to a spacer molecule; a plurality of spacer molecules attached at a first end to said surface in each of the discrete areas, each of said spacer molecules adapted to being attached at its second end to a metallic surface or a label, each of said spacer molecules having a site between its first end and its second end capable of being cleaved; a first n-mer oligonucleotide having a first sequence attached to substantially all of the spacer molecules between the cleavage site and the first end of the spacer molecule, and a second n-mer oligonucleotide having a second sequence attached to substantially all of the spacer molecules; wherein substantially no other discrete areas on the surface of the substrate contain spacer molecules having n-mer oligonucleotides having the first sequence attached thereto and n is an integer selected from the integers 4-10,000, most advantageously 6-28.
The present invention also encompasses a method for determining the sequence of a (p+q+r)-mer segment of a gene suspected of being present in a sample comprising: (a) forming a solution of the sample and a mixture of q-mer oligonucleotides having all possible sequences of a q-mer oligonucleotide, or, optionally, a subset of all such possible sequences; (b) contacting an assay element with at least a portion of the solution of step (a), the assay element having a surface and plurality of spacer molecules bound to the surface, the spacer molecules having a first end bound to the surface and a second end bound to a metallic surface or a label and a cleavage site intermediate between the first and second ends, the spacer molecules further having a first p-mer oligonucleotide attached thereto between the cleavage site and the first end and a second r-mer oligonucleotide attached thereto between the cleavage site and the second end, the combination of p-mers and r-mers including all combinations of oligonucleotide sequences of p-mer and r-mer oligonucleotides, or, optionally, a subset of all such combinations, each particular combination of sequences of the p-mer and r-mer oligonucleotides being at a predetermined location on the surface; (c) ligating the resultant hybridized oligonucleotides attached to the spacer molecules resulting from step (b) above; (d) detecting the presence or absence of a particular sequence combination of the hybridized oligonucleotides at each predetermined location on the surface; and (e) processing the sequence information obtained from step (d) to deduce the sequence of the (p+q+r)-mer oligonucleotide present in the sample, wherein p, q and r are integers selected from the integers 4-10,000, most advantageously 6-26, and (p+q+r) does not exceed 30,000, and most advantageously 60. Steps (a)-(e) can be performed in parallel for different, multiple segments of a gene.